In-vivo energy depleting strategies for killing drug-resistant cancer cells

ABSTRACT

This invention also provides a method for treating a cancer subject comprising administering to the subject a combination of ATP-depleting agents at concentrations which deplete the ATP level to, or close to, at least 15% of normal in cancer cells wherein at least one of the ATP-depleting agents is a mitochondrial ATP-inhibitor, a methylthioadenosine phosphorylase inhibitor or an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside, wherein said composition produces a substantially better effect than a composition without at least one of the ATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor and an inhibitor of De Novo purine synthesis other than 6-Methylmercaptopurine riboside.

This application is a continuation-in-part of U.S. Ser. No. 10/172,346,Filed 13 Jun. 2002, the content of which is hereby incorporated intothis application by reference.

The invention disclosed herein was made with government support underNational Cancer Institute RAID Grant Application #153. Accordingly, theU.S. Government has certain rights in this invention.

Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION AND CANCER RELEVANCE

Drug resistance is the central problem of cancer chemotherapy.Clinically effective combination chemotherapy can cause impressiveobjective tumor response rates, including complete tumor regressions,but some cancer cells are of lesser sensitivity to the agent (i.e., aredrug-resistant), are only damaged, recover, and re-grow. The delayedtumor recurrence yields only a short remission period with littleimprovement in survival time.

There are many mechanisms of drug resistance. Multiple independentmechanisms of drug resistance may coexist in a population of tumor cellsas well as in the same cancer cells, as they arise from multiple geneticchanges in single cell clones, and are part of the heterogeneity of theneoplastic process. Mechanisms of drug resistance have been largelyidentified, and this knowledge has suggested many specific approaches toovercoming one or another type of clinical drug resistance, but theseattempts have failed as they also potentiate drug toxicity towardsnormal tissues.

The therapeutic research strategy for several decades has been that theadministration of multiple drugs with different properties andmechanisms of action at optimal doses and intervals should result incells resistant to one class of drug being killed by another drug in theregimen. However, the extensive, clinical data over these decades hasevidenced only a minor impact on treatment outcome along withtroublesome and serious toxic side effects (e.g., emesis, diarrhea,alopecia, asthenia, fatigue, myelosuppression, febrile neutropeniarequiring hospitalization, neurosensory and neuromotor disturbances,arthralgias and myalgias, heart failure, and treatment-related deaths).Despite the long dismal history of repeated failures to meaningfullyimprove survival rates by aggressive combination chemotherapy withnon-cross-reacting drugs, hope is nevertheless expressed that the futurewill be different with the new molecularly targeted agents.

However, no matter how many effective mechanistically-differentanticancer agents there are, and no matter how superior theirtherapeutic index, cancer cell demise occurs by only two cell deathpathways (necrosis or apoptosis). If the two cell death mechanisms areattenuated by drug resistance mechanisms (e.g. p-glycoprotein and/orglutathione prevent intracellular drug levels reaching concentrationlevels sufficient to fully activate the necrosis pathway; caspasedeletions and endogenous caspase inhibitors prevent completion ofapoptosis), these tumor cells are only sublethally injured, recover, andproliferate to kill the patient. The history of the results of theseclinical trials is therapeutic equivalence between different combinationchemotherapy “doublets” and “triplets”. The repeated failures of thisapproach to overcoming clinical drug resistance—i.e., the lack ofclinically relevant differences in overall survival-means onlycontinuance of palliative treatment with decisions tailored individuallyaround such issues as differences in toxicity profiles, patients' ageand performance status, and quality of life.

New agents, no matter a new molecular target or superior therapeuticindex, can only kill cancer cells if there is completion of the celldeath pathways through death's door. In drug-resistant cells, it is notthe activation of their cell death pathways by clinically effectiveanticancer agents that is at fault, but rather pathway completion todeath of the cell. This reality suggests that continuance of this failedstrategy utilizing only aggressive combination chemotherapy withnon-cross-reacting drug-will likely result in-to quote Yogi Berra—“déjàvu all over again”.

The above facts suggest the development of a treatment (co-administeredwith the initiation of activity in the cell death pathways by anticanceragents) that complements and augments the agent-induced initiation ofactivity in cell death pathways to bring the pathway to completion;namely, to cell death. That treatment, focused on severe ATP depletion,has been developed and proven at the preclinical level, and is about toundergo validation by clinical trial with clinical supplies of theATP-depleting regimen provided by the NCI RAID grant mechanisms.

Heterogeneous neoplastic cell populations likely contain cancer cells ofvariable sensitivity to the anticancer agents. Less sensitive cellswould not receive enough damage to reduce ATP to low levels sufficientto cause necrotic death. We hypothesized that biochemical modulation tofurther depress ATP to lower lethal-inducing levels would kill thesesublethally-injured cells, augment tumor regressions, and perhaps evenyield some cures.

SUMMARY OF THE INVENTION

This invention provides a composition comprising a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells, wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

The ATP level is depleted to at least 15% of normal in cancer cells. Insome embodiments, it is substantially lower than 15%. For example, thelevel could be 5% of normal. In a further embodiment, the level is aslow as 1%.

This invention also provides a composition comprising an effectiveamount of a combination of ATP-depleting agents at concentrations whichdeplete the ATP level to at least 15% of normal in cancer cells, and apyrimidine antagonist, wherein at least one of the ATP-depleting agentsis a mitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof.

This invention also provides a method for treating a cancer subjectcomprising administering to the subject a combination of ATP-depletingagents at concentrations which deplete the ATP level to, or close to, atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof.

This invention also provides a method for treating a cancer subjectcomprising administering to the subject a combination of ATP-depletingagents at concentrations which deplete the ATP level to, or close to, atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, wherein said composition produces a substantiallybetter effect than a composition without at least one of the followingATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

This invention also provides a method for induction of cancer cell deathcomprising contacting said cancer cell with a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof.

This invention also provides a method for induction of cancer cell deathcomprising contacting said cancer cell with a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, wherein said composition produces a substantiallybetter effect than a composition without at least one of the followingATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

This invention provides a method for treating a cancer subject, and forthe induction of cancer cell death, comprising administering to thesubject a combination of ATP-depleting agents, a pyrimidine antagonist,and anticancer agent to which the treated cancer is sensitive, atconcentrations which together collectively deplete the ATP levels to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitors, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

This invention provides a method for treating a cancer subject, and forthe induction of cancer cell death, comprising administering to thesubject a combination of ATP-depleting agents, a pyrimidine antagonist,and anticancer agent to which the treated cancer is sensitive, atconcentrations which together collectively deplete the ATP levels to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, wherein said composition produces an effect whichis substantially better than a composition without at least one of thefollowing ATP-depleting agents: a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, aninhibitor of De Novo purine synthesis other than 6-Methylmercaptopurineriboside, or a combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

Patent Cooperation Treaty (PCT) Application PCT/US01/46886(International Publication Number WO 02/4720A1) discloses treatment ofcancer by reduction of intracellular energy and pyrimidine.Specifically, PCT/US01/46886 highlighted the importance of depletion ofATP in cancer therapy. The disclosure herein provides improvements overthis PCT application.

This invention provides a composition comprising a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells, wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

The ATP level is depleted to at least 15% of normal in cancer cells. Insome embodiments, it is substantially lower than 15%. For an example,the level could be 5 to 10% of normal. In another instance, the level is1 to 4%. In a further embodiment, the level is as low as 1%.

This invention also provides a composition comprising an effectiveamount of a combination of ATP-depleting agents at concentrations whichdeplete the ATP level to at least 15% of normal in cancer cells, whereinat least one of the ATP-depleting agents is a mitochondrialATP-inhibitor, a glycolytic inhibitor, a methylthioadenosinephosphorylase inhibitor, an inhibitor, of De Novo purine synthesis otherthan 6-Methylmercaptopurine riboside, or a combination thereof.

This invention also provides the above compositions, wherein saidcompositions produce a substantially better effect than a compositionwithout at least one of the following ATP depleting agents: amitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof.

As used herein, substantially better means that the composition coulddeplete the intracellular energy level at least 5% better. In anotherembodiment, the composition is 5% to 100% better. In a separateembodiment, it is 10% to 100% better. In a further embodiment, it is 15%to 100% better. In a still further embodiment, the composition is 20% to100% better. In a further embodiment, it is 25% to 100% better. Inanother embodiment, it is 30% to 100% better. In a separate embodiment,it is 35% to 100% better. In another embodiment, it is 40% to 100%better. In a further embodiment, it is 45% to 100% better. In anotherembodiment, it is 50% to 100% better. In a still further embodiment, itis 55% to 100% better. In a separate embodiment, it is 60% to 100%better. In a still separate embodiment, it is 65% to 100% better. Inanother embodiment, it is 70% to 100% better. In a separate embodiment,it is 75% to 100% better. In another embodiment, it is 80% to 100%better. In a separate embodiment, it is 85% to 100% better. In a furtherembodiment, it is 90% to 100% better. Finally, the composition may be95% to 100% better.

To achieve “substantially better than the composition without”, it isnot necessary that the depleting agents used be more than one agent. Infact, a single agent may be capable of performing such better effect.

The above compositions may further comprise a pyrimidine depletingagent. Further, the above compositions may comprise an anticancer agentto which the cancer is sensitive. In a still further embodiment, theanticancer agent is at approximately half the maximum tolerated dose.

These agents stated hereinabove may be a single agent but with more thanone function. For example, an ATP-depleting agent may also be ananticancer agent.

The ATP-depleting agents include but are not limited to6-methylmercaptopurine riboside (MMPR), 6-Aminonicotinamide (6-AN) oralanosine (AL).

The above compositions may further comprise N(phosphonacetyl)-L-asparticacid (PALA). In a separate embodiment of the compositions, it comprises3-bromopyruvic acid.

The above composition further comprises dehydroepiandrosterone (DHEA),oxythiamine (OT) or in combination thereof.

In an embodiment, the composition further comprises 6-Aminonicotinomide(6-AN).

The above composition may further comprise a cytokine. In an embodiment,the cytokine is G-CSF.

This invention also provides a pharmaceutical composition comprising theabove composition and a pharmaceutically acceptable carrier.

For the purposes of this invention, “pharmaceutically acceptablecarriers” mean any of the standard pharmaceutical carriers. Examples ofsuitable carriers are well known in the art and may include, but are notlimited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution and various wetting agents. Othercarriers may include additives used in tablets, granules and capsules,etc. Typically such carriers contain excipients such as starch, milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gum,glycols or other known excipients. Such carriers may also include flavorand color additives or other ingredients. Compositions comprising suchcarriers are formulated by well-known conventional methods.

This invention also provides the combination of ATP-depleting agents andthe anticancer agent to treat drug-resistant cancer cells. In anembodiment, the dose of the anticancer agent is at approximately half ofthe maximum tolerated dose.

This invention also provide a method for treating a cancer in a subjectcomprising administering to the subject a combination of ATP-depletingagents at concentrations which deplete the ATP level to, or close to, atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside orcombination thereof.

This invention also provides a method for treating a cancer subjectcomprising administering to the subject a combination of ATP-depletingagents at concentrations which deplete the ATP level to, or close to, atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitor, methylthioadenosine phosphorylase inhibitor, an inhibitor ofDe Novo purine synthesis other than 6-Methylmercaptopurine riboside, ora combination thereof, and said composition produces a substantiallybetter effect than a composition without at least one inhibitor of thefollowing ATP-depleting agents: a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, aninhibitor of De Novo purine synthesis other than 6-Methylmercaptopurineriboside or a combination thereof.

This invention also provides the above methods wherein the agentsfurther comprise a pyrimidine-depleting agent.

The above methods may further comprise an anticancer agent. In anotherembodiment, the cancer is clinically sensitive to the employedanti-cancer agent.

In a separate embodiment, the anticancer agent is at approximately halfof the maximum tolerated dose.

This invention also provides a method for induction of cancer cell deathcomprising contacting said cancer cell with a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof.

This invention also provides a method for induction of cancer cell deathcomprising contacting said cancer cell with a combination ofATP-depleting agents at concentrations which deplete the ATP level to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, wherein said composition produces a substantiallybetter effect than a composition without at least one of the followingATP-depleting agents: a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

The above methods may comprise a pyrimidine-depleting agent. In anembodiment, the above methods further comprise an anticancer agent.

In an embodiment, the cancer is clinically sensitive to the employedanticancer agent. In a further embodiment, the anticancer agent is atapproximately half of the maximum tolerated dose.

This invention provides a method for treating a cancer subject, and forthe induction of cancer cell death, comprising administering to thesubject a combination of ATP-depleting agents, a pyrimidine antagonist,and anticancer agent to which the treated cancer is sensitive, atconcentrations which together collectively deplete the ATP levels to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitors, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

This invention provides a method for treating a cancer subject, and forthe induction of cancer cell death, comprising administering to thesubject a combination of ATP-depleting agents, a pyrimidine antagonist,and anticancer agent to which the treated cancer is sensitive, atconcentrations which together collectively deplete the ATP levels to atleast 15% of normal in cancer cells wherein at least one of theATP-depleting agents is a mitochondrial ATP-inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside, or acombination thereof, wherein said composition produces an effect whichis substantially better than a composition without at least one of thefollowing ATP-depleting agents: a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor, aninhibitor of De Novo purine synthesis other than 6-Methylmercaptopurineriboside, or a combination thereof.

In an embodiment of the above methods, the anticancer agent is atapproximately half of the maximum tolerated dose.

The ATP-depleting agents used in the above methods include but are notlimited to 6-methylmercaptopurine riboside (MMPR), 6-Aminonicotinamide(6-AN) and alanosine (AL).

In an embodiment of the above methods, the methods further compriseN-(phosphonacetyl)-L-aspartic acid (PALA).

In an embodiment of the above methods, the method further comprises3-bromopyruvic acid.

In an embodiment of the above methods, the method further comprisesN-(phosphonacetyl)-L-aspartic acid (PALA).

In an embodiment of the above methods, the method further comprisedehydroepiandrosterone (DHEA), oxythiamine (OT) or in combinationthereof.

In an embodiment of the above methods, the method further comprises6-Aminonicotinamide (6-AN).

In an embodiment of the above methods, the method further comprises acytokine. The cytokine includes but is not limited to G-CSF.

This invention also provides a method for killing drug-resistant cancercells comprising contacting the combination of ATP-depleting agents andthe anticancer agent. In an embodiment, the dose of the anticancer agentis at approximately half of the maximum tolerated dosage.

This invention provides a method for treating drug-resistant cancercells comprising contact the said cancer with a combination ofATP-depleting agents and an anticancer agent.

In an embodiment the dose of said anticancer agent is at approximatelyhalf of the maximum tolerated dose.

This invention provides the above method, wherein the ATP level isdepleted to at least 15% of normal in cancer cells and at least one ofthe ATP-depleting agents is a mitochondrial ATP-inhibitor, a glycolyticinhibitor, a methylthioadenosine phosphorylase inhibitor, an inhibitorof De Novo purine synthesis other than 6-Methylmercaptopurine riboside,or a combination thereof.

This invention further provides the above described method wherein theATP level is depleted to at least 15% of normal in cancer cells and atleast one of the ATP-depleting agents is a mitochondrial ATP-inhibitor,a glycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor,an inhibitor of De Novo purine synthesis other than6-Methylmercaptopurine riboside, or a combination thereof, within andsaid composition produces an effect which is substantially better than acomposition without at least one of the ATP-depleting agents: amitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor, an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside or acombination thereof.

Finally, this invention provides method for induction of cancer celldeath comprising contacting said cancer cell with an agent capable ofinducing necrosis in cancer cells. In an embodiment the agent is anATP-depleting agent. This method may include a pyrimidine-depletingregimen, or a combination thereof.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

Experimental Details

MAP

An ATP- reducing regimen, MAP - - - an acronym for the combination of6-methylmercaptopurine riboside, MMPR, +6-aminonicotinamide, 6-AN,+N-(phosphonoacetyl)-L-aspartic acid, PALA) - - - when co-administeredwith anticancer agents; e.g., Doxorubicin A (Adriamycin) - - - enhancesexperimental tumor regressions in vivo via the necrosis cell deathpathway (1). MMPR is an inhibitor of de novo purine biosynthesis thatlimits adenine supplies for ATP production. (2) 6-AN is an inhibitor ofthe generation of ATP in the glycolysis pathway (3-6). The cell-killingATP threshold is 15% of basal levels, and lower (7-8). Since multiplebiochemical pathways contribute to the synthesis, generation andmaintenance of ATP, a combination of two ATP-depleting agents,MMPR+6-AN, is co-administered with effective anticancer agents toconcomitantly block several of the ATP- producing pathways to achievethe ATP- depleting objective of ≦15% of normal. PALA, a de novopyrimidine biosynthesis inhibitor, can, at low non-toxic dosages,selectively lower pyrimidine nucleotide levels in tumors (9). MMPR+6-ANcan markedly lower ATP levels to an average of 15% of normal in murinemammary cancers (1). In the presence of such severely depleted ATPlevels the salvage pathway formation of pyrimidine di-and tri-phosphatescan be inhibited at the kinase step. Biochemical analysis shows thatMAP-treated tumors are severely depleted of two vitally neededmetabolites: the UTP pools to 14% of normal (10), and ATP to 15% ofnormal (1).

Necrosis cell death ensues because intracellular ATP levels at 15% ofbasal or lower can not sustain cellular homeostasis (7-8, 11-12).Indeed, intracellular ATP levels determine cell fate by apoptosis ornecrosis (11) and can effect a “switch” in the cell death mode betweenapoptosis and necrosis (12). There are many references that anticanceragents that usually cause cell death by apoptosis, when blocked byexogenous or endogenous caspase inhibitors, or by manipulation (i.e.,depletion) of energy metabolism, “switch” to necrosis cell death due tosevere ATP depletion (references summarized in Table 1). TABLE 1EVIDENCE IN NON-DRUG RESISTANT CELLS THAT APOPTOTIC INDUCERS CAN BE MADETO “SWITCH” TO THE NECROSIS CELL DEATH PATHWAY Apoptotic Apoptosis NoInducer Cells Blocker Result Reference 1 EGF Pancreas Caspasel-Apoptosis to FEBS inhibitor non-apoptotic Lett. ‘necrotic’- 491:108-108,like deaths 2001 2 Staurosporin, Jurkat Inhibitor of Apoptosis to CellDeath VP-16, Act Lymphoma caspases necrosis Differ. D T-cell 5:298-306,1998 3 TNF L929 and Inhibitor of Enhanced J. Exp. HeLa cells caspasesnecrosis Med, 187:1477-1485, 1998 4 Staurosporin, Human T Pre-emptiedApoptosis to Cell Death CD95 cells cells of ATP necrosis Differ.stimulation 4:435-442, 1997 5 Fas mAb Jurkat T ATP Apoptosis to Cancerstimulation cells, HeLa depletion necrosis Res. 57:1835-1840, 1997 6Dexamethasone B-lymphocytes Inhibitor of Apoptosis to FEBS Lett.caspases necrosis 425:266-270, 1998 7 Dexamethasone Thymocytes Inhibitorof Apoptosis to Oncogene Etoposide caspases necrosis 15:1573-1581, 19978 Staurosporin, Human T Per-emptied Apoptosis to J. Exp. CD95 agonistcells cells of ATP necrosis Med. 185:1481-1486, 1997 9 Deoxycholic HCT116 Overexpression Apoptosis to Cancer acid cells on of bcl-2 necrosisLett. (activates or PKC 152:107-113, Fas) 2000 10 H₂O₂ HN U937Increasing Apoptosis to Exp. Cell cells H₂O₂ necroses Res concentration221:462-469, 1995 11 Bax Jurkat cell Inhibitor of Apoptosis to Proc Natlcaspases “non- Acad Sci. apoptotic” 93:14559, death 1996 12 CamptothecinLeukemia U- Inhibitor of Apoptosis to Cancer 937 cells caspases necrosisRes. 59:3565-3569, 1999 13 Anti-F25 L 929 cells Inhibitor of Apoptosisto J. Exp. Abs caspases necrosis Med. 188:919-930, 1998 14 Anti-F25Jurkat Inhibitor of Apoptosis to J. Cell Abs cells caspases necrosisBiol. 143:1353-1360, 1998 15 TNF-a Mouse 3T3 Inhibitor of Normally J.Virol. fibroblasts caspases resistant; 74:7470-7477, switch to 2000necrosisThe reason is that anticancer agents usually damage DNA, either directlyor indirectly, and thereby activate the two principal pathways of celldeath, necrosis and apoptosis, simultaneously in the same cancer cell.Only one death pathway prevails in the cells, as determined byintracellular conditions. However, the two modes of cell death can occurin different cells simultaneously in the same tumor exposed to the sameanticancer agent. One reason is that different drug concentrations reachdifferent cancer cells; low concentrations induce apoptosis and highconcentrations cause necrotic cell death (13). If the anticancer drug'sintracellular concentration is high, the drug-DNA “hit” is then ofsufficient magnitude to strongly activate poly (ADP-ribose) polymerase(PARP), and PARP rapidly destroys and depletes glycolytic NAD severelyinhibiting the glycolytic production of ATP (1). The resulting severedepletion of ATP causes cell death by necrosis (1). And, although lowconcentrations of anticancer agents do not damage DNA sufficiently tocause the marked lowering of ATP that results in necrotic cell death,there is still a reduction of ATP. Hence, all effective anticanceragents always cause ATP reduction, and are considered part of ourATP-depleting regimen. An important part, because as “selective” agentsfor cancer cells, effective anticancer agents preferentially reduce moreATP in cancer cells than normal cells. The agents thereby create atherapeutic opportunity for co-administration of biochemical modulators(i.e., the concomitant addition of an ATP-depleting regimen-e.g., MAP)to further reduce cancer cell ATP to the severely low levels (i.e., ≦15%of normal) that kill, and the cells die a necrotic death.

MAP plus each of ten mechanistically different anticancer drugs athalf-MTD (doxorubicin, paclitaxel, docetaxel, cisplatin, phenylalaninemustard, mitomycin C, cyclophosphamide, etoposide, 5-fluorouracil, andradiotherapy) has been demonstrated in vivo to be safe and to causesignificantly greater tumor-regressions than the MTD of each anticanceragent alone in hundreds of animals bearing advanced spontaneous andfirst generation murine breast cancer (references summarized in 1), aswell as in murine leukemia and colon tumors. “Cures” (25%) were producedwith MAP+radiotherapy in tumor-bearing mice followed for over 380 days(14). The improved therapeutic results were all produced by MAP+eachanticancer agent at half-MTD, strongly suggesting that a markedreduction of the toxic side effects of usual MTD clinical cancerchemotherapy will be obtained in future clinical trials.

Spontaneous murine breast cancers have drug-resistant cancer cells aspart of their heterogeneous neoplastic cell population just asspontaneously-arising human cancers do. The fact that the number oftumor regressions induced by the combination of MAP+ anticancer agentswere always significantly greater than the regressions produced by eachanticancer agents alone at MTD suggested that the additional cell killsoccurred in drug-resistant cancer cells. Drug-resistant cancer cells areknown to be frequently blocked in apoptosis, but not known to haveblocks per se in the necrosis cell death pathway. It is likely thatdrug-resistant cells were killed in the spontaneous tumor because theATP-depleting strategy is specifically targeted at the latter pathway.However, to obtain a clearer focus of the effect of the ATP-depletingstrategy plus an anticancer agent on drug-resistant cancer cells,treatment was done on multi-drug-resistant, overexpressed p-glycoproteinNCI-AR-Res (Adriamycin-resistant) human mammary carcinoma xenografts(15). (See Table 2, below)

MAPAL

As previously noted, the ATP depletion induced in murine mammary tumorsby (MMPR+6-AN) of MAP averaged 15% of normal, the cytocidal thresholdlevel of ATP shown to be insufficient to sustain cell viability (7-8).Hence, some individual tumor cells have ATP levels <15% of normal, arekilled, and the tumors regress. Other MAP-treated tumors have reducedATP levels >15% of normal and these tumors are only inhibited in theirgrowth (7). Obviously if the (MMPR+6-AN) induced ATP tumor level hadaveraged lower than 15% of normal, there would be more tumor cellskilled, and more tumor regressions. A stronger ATP-depleting regimen wastherefore sought. Alanosine (AL), an inhibitor of de novo adenosinemonophosphate synthesis (16), had been previously shown to add to MMPR'sreduction of ATP (17). In the latter studies, MMPR alone lowered ATPlevels to 49% of normal, and alanosine (AL) alone to 63% of normal, butin combination they decreased ATP to 34% of normal. This strikingsynergistic reduction of ATP when combined suggested that the additionof AL to MAP(acronym: MAPAL) might safely reduce the average tumor ATPlevels still lower than the average 15% of normal induced by MAP. Wehave not the funding opportunity to compare in the same tumors theaverage % of induced ATP depletion by MAPAL as compared to MAP. However,the evaluation of MAPAL+an antitumor agent to MAP+the same anticanceragent against advanced mammary tumors in mice showed MAPAL to betherapeutically more effective than MAP, and with no mortality and onlyan 8% weight loss (unpublished studies).

MAPAL, therefore, was the ATP-depleting regimen chosen to be evaluatedagainst NCI-AR-RES (Adriamycin-resistant) xenografts, and thetherapeutic results are summarized in Table 2. TABLE 2 THE ADDITION OFAN ATP-DEPLETING REGIMAN, MAPAL, ENABLES LO-DOSE ADRIAMYCIN TO KILLDRUG-RESISTANT CANCER CELLS IN ADRIAMYCIN-RESISTANT HUMAN MAMMARYCARCINOMA XENOGRAFTS BY THE NECROSIS CELL DEATH PATHWAY Treatment BodyWt p- Group^(a) n^(c) change^(c) % PR^(c) % Value^(d) 1. Control 20 +6.6 0 2. Adria₁₀ 57 −7.8  0 3. MAPAL^(b) 76 −3.8 18 (14/76) 0.0003 4. MAPAL→ 77 −7.9 48 (37/77) <.0001    2½ hr→ Adria₅ 5. MAPAL + Adria₅ 52 −9 46(24/52) <.0001    (SIMULTANEOUSLY)^(a)Pooled experiments (99, 103, 108, 112, 114, 115, 116, 117): Nudemice (nu/nu) with NCI-AR-RES mmamry carcinoma xenografts averaged 100 mgwhen treatment initiated. Indicated treatment administered in threecycles with a 14 day interval between cycles.^(b)MAPAL = 6-methylmercaptopurine riboside (MMPR₁₅₀) +6-aminocotinamide (6-AN₆) + PALA₁₀₀ + alanosine (AL₂₅₀); subscript =mg/kg. PALA 17 hrs prior to (MMPR + 6 − AN + AL) 2.5 hrs prior to Adria.All agents i.p., except Adria i.v.^(c)Therapeutic observations recorded one week after the third cource(35 days after initiating therapy.). PR = ≧50% decrease in tumor volume^(d)Compared to Adria₁₀Group 2. Comparing Group 3 VS Group 4 =<.0001; VSGroup 5, 0.0011

Comment—The combination of MAPAL and Adria (48% PR or 46% PR) is moreeffective in killing cells than just MAPAL (18% PR), and the use of onlyhalf- MTD of Adria in the presence of MAPAL (48% PR or 46% PR) isremarkably more effective in killing drug-resistant cancer than the fulldose(MTD) of Adria (0% PR). The striking positive therapeuticresults—almost a 50% P.R. rate using a 50% lower dose of Adria (withMAPAL) compared to a 0% P.R. rate with high dose Adria(alone)—auger wellfor an amelioration of the toxic side effects of high dose Adria inclinical trial along with greater therapeutic results.

It is stressed that there is no difference between the PR rates of Group4, MAPAL 2½ hours prior to Adria, vs. Group 5, MAPAL simultaneouslyadministered with Adria; namely, 48% P.R. vs. 46% P.R., respectively. Itis therefore clear that the time differential (2½ hrs.) did not affectthe results; suspicion had been raised that the ATP-dependentp-glycoprotein drug efflux pump might be adversely affected by prior(2½hr) ATP reduction in Group 4 as the reason for previously publishedpositive therapeutic results with that time interval.

Molecular biology studies (Table 1) demonstrate that the necrosis celldeath pathway can fully function (i.e., kill) to bypass a blockedapoptosis pathway, or can be fully evoked following manipulation ofenergy metabolism to kill cells. The findings in these studies innon-drug resistant cells appear relevant to drug-resistant cells, whichare blocked in apoptosis but not known to have blocks per se in thenecrosis cell death pathway. Thus, if their apoptosis pathway is blockedbut their necrosis pathway is “open”, a combination of anticancer agentsand ATP-depletion agents should be able to lower ATP to cytocidal levelsand selectively kill drug-resistant cells. The findings in these fifteenreferenced studies imply that the necrosis cell death pathway could bethe therapeutic opportunity to overcome drug-resistant cancer cells.

The primary similarity in the treated cancer cells of these molecularbiology studies to drug-resistant cells is that apoptosis activity isprevented in both. In the drug-resistant cells triggered apoptosiscannot proceed to completion because of inherent multiple drugresistance factors; e.g., endogenous caspase inhibitors (so-calledIAPs), genetic deletions of caspases, over-expressed anti-apoptoticbcl-2, mutations in p53 and other processing units in the apoptoticprogram, or in some individual cancer cells the presence of all of thesegenetically-induced resistance factors. Thus, apoptosis may be triggered(initiated), but not completed, by drug-treatment in the manyabove-noted molecular biology studies as well as in the drug-resistantcancer cells.

However, there is a significant difference in comparing necrosispathways. In the molecular biology studies with non-drug resistant cellsthere are no resistance factors (i.e., obstacles) leading to thenecrosis pathway, and the usual apoptosis cell death mode is readily“switched” to necrosis. In contrast, drug-resistant cells can haveresistance factors (e.g., drug efflux enzymes) that only allow for lowdrug concentrations that effect only partial completion of the necrosispathway). High intracellular drug concentrations are required for celldeath by necrosis. Necrosis will not occur unless the drug-target “hit”(usually to DNA) is of sufficient intensity to strongly activate PARP soas to result in a rapid and severe depletion of ATP to cell-killinglevels (≦15% of normal). In drug-resistant cancer cells, thedrug-induced necrosis pathway is initiated but does not proceed tocompletion (i.e., to death) because the drug-resistance factors ofover-expressed drug efflux pumps (e.g., p-glycoproteins), and/orintracellular drug detoxifiers (e.g., glutathione), lower theintracellular drug level and diminish the drug-target interaction.Hence, although the result is still reduction of ATP, it is not lowenough to be lethal-inducing (i.e., ≦15% normal ATP). The necrosispathway is only partially activated, and the ATP depletion does not fallto levels sufficient for cell-killing. Reductions of ATP to above thecell-killing threshold will only cause transient inhibition of cancercell proliferation followed by complete recovery (7). Thus, followingpresent chemotherapy, drug-resistant cancer cells are onlysub-lethally-injured, recover, and grow back to kill the patient.

The molecular biology references (Table 1) suggest that a substantialbut non-lethal degree of ATP depletion always occurs in anticanceragent-induced sub-lethally-injured drug-resistant cancer cells becausethere is only initiation, but not completion (i.e., to death) of thenecrosis cell death pathway. Thus, since most effective DNA-damaginganticancer agents are themselves ATP-reducing agents, the furtherreduction of ATP to the ATP lethality threshold by an ATP-depletingregimen should kill these drug-resistant cells - - - and does (Table 2).Previous preclinical studies successfully overcoming one or another ofthe many drug-resistant factors have foundered in the clinical arenabecause drug-resistant factors are multifactorial. However, the novelATP-depleting strategy is not aimed at any of the drug-resistantfactors, but bypasses them all by directly killing drug-resistant cancercells through their vulnerable necrosis cell death pathway.

PALM-Bromopyruvate(BrPA)

In non-drug-resistant tumors, Geschwind et al (18) have reported that apotent ATP-depleting agent (3-bromopyruvic acid, or BrPA), whendelivered by “direct” intraarterial administration to liver-implantedrabbit tumors, eradicates nearly all viable cancer cells by necrosis,and also without harm to normal cells or to the animals. The absence ofmortality and serious toxicity further obviates the notion that severeintra-tumor ATP depletion must be toxic to both normal and cancer cells.

No ATP measurements were reported in the successful cancer cell-killingtherapy with BrPA as a single agent. The very high concentration of BrPAachieved by the “direct” arterial delivery apparently depleted ATP inthe “directly”-treated cancer cells to cell-killing ATP levels becausethe same dose of BrPA by systemic i.v. administration only inhibitedtumor growth, apparently due to a diluted concentration by the wholeblood volume only lowering ATP to levels above the cell-killing ATPthreshold, levels shown to effect only inhibition of tumor growth. Thelevel of ATP depletion is all important as regards the therapeuticresult (ie, death of cells vs. their transient inhibition). Thisunderstanding provides insight for the past failures and present successof ATP-depletion.

Previous attempts at ATP-depletion as therapy have failed due to thelack of understanding of how it would be effective in killing cancercells. Only recently has there been data establishing that there is acell-killing ATP threshold of 15% of basal levels and below(7-8), andthat attaining this severe lowering of intracellular ATP levels incancer cells normally requires the concomitant administration ofmultiple ATP-depleting agents(1).

In line with our planned step by step development of a “best”multiple-ATP-depleting regimen, the incorporation of “selective”antimitochondrial agents (e.g., F16; 19) had been programmed foreventual inclusion in the ongoing development of the new and promisingATP-depleting therapeutic strategy. Although BrPA has not been reportedas “selective”, it was noteworthy in reducing cellular ATP productionvia the inhibition of both glycolysis and oxidative phosphorylation(18). It was therefore substituted for the glycolysis inhibitor, 6-AN,in MAPAL. Without 6-AN, MAPAL includes only PALA+Alanosine (AL)+MMPR(acronym: PALM). PALM+BrPA, or PALM-BrPA, inhibits adenine biosynthesis,glycolytic and mitochondrial ATP production. PALM-BrPA+Adria₅ vsMAPAL+Adria₅ were compared in therapeutic studies inmulti-drug-resistant NCI-AR-RES (Adriamycin-resistant) human mammarytumor xenografts. Table 3 pools the data of four experiments. TABLE 3PALM-BRPA IS SIGNIFICANTLY SUPERIOR TO MAPAL IN ENHANCINGADRIAMYCIN-DRUG-RESISTANT CANCER CELL KILL. PALM-BRPA INDUCES SEVERE ATPREDUCTION (BY INHIBITING ADENINE BIOSYNTHESIS, GLYCOLYTIC ANDMITOCHONDRIAL ATP PRODUCTION) THAT ENABLES LO-DOSE ADRIAMYCIN TO KILLADRIAMYCIN-RESISTANT CELLS IN HUMAN MAMMARY XENOGRAFTS BY THE NECROSISCELL DEATH PATHWAY. Treatment Body Wt Deaths^(c) PR^(c) Group^(a) n^(c)change^(c) % % % p-Value 1. Control 8 +5 0 2. Adria₁₀ 15 −6 0 3. MAPAL +Adria₅ 41 −8 4 34 4. PALM + BrPA₁₀ ₊ 42 −13 0 62 ^(d 0.01%)    Adria₅^(a)Four pooled experiments (116, 117, 121, 122): Nude female mice withNCI-AR-Res human mammary carcinoma xenografts averaging 100 mg whentreatment initiated. Indicated treatments were administered in 3 cycleswith a 14 day interval between cycles.^(b)MAPAL = MMPR₁₅₀ + 6-AN₆ + PALA₁₀₀ + AL₂₅₀. PALM = PALA₁₀₀ + AL₂₅₀ +MMPR₁₅₀. BrPA = 3-Bromopyruvate₁₀. All agents i.p., except BrPA andAdria. i.v. Subscripts = mg/kg.^(c)Therapeutic observation one week after the third course (35 daysafter initiating therapy). PR = ≧50% reduction in tumor size compared totumor size when treatment initiated. PR % = number PR per group ÷surviving animals per group × 100.^(d)Compared to Group 3, MAPAL + Adria₅.

Comment-As previously (Table 2), the MTD of Adria, 10 mg/kg, on thisAdriamycin-resistant tumor is without tumor-regressing effect (0% PR),whereas Adria. at ½ MTD in the presence of MAPAL causes an impressivenumber of partial tumor regressions in drug-resistant cells (34% PR),and PALM-BrPA+Adria(½ MTD) is remarkably much more effective (62% PR).With only these four experiments completed, the PALM-BrPA data isconsidered preliminary. Nevertheless, PALM-BrPA+Adria₅ appears to havedoubled (62%) the PR rate over MAPAL+Adria (34%). We have not had thefunding opportunity to compare the degree of ATP depletion. Bothcombinations inhibit adenine supplies (i.e., by MMPR), both inhibitadenosine monophosphate biosynthesis (i.e., by AL), and both areglycolytic inhibitors, although by different mechanisms - - - PALM-BrPAby BrPA, and MAPAL by 6-AN. Although one may be a slightly strongerglycolytic inhibitor, the major difference clearly is that onlyPALM-BrPA is also a mitochondrial ATP inhibitor. Therefore, it could beanticipated that PALM-BrPA would be the stronger ATP depleter, and thetherapeutic results confirm this expectation.

MAP with two ATP depleters effected a tumor ATP average to the cytocidalATP threshold of 15% of normal (1). MAPAL with three ATP depletersapparently further reduced the average ATP level below this criticalcytocidal ATP threshold level. PALM-BrPA with four ATP depletersapparently reduced this critical ATP average still more. Thus, with eachincremental decrease in ATP depletion below the cytocidal threshold of15% of normal, more drug-resistant cancer cells are killed.

Combining a number of ATP-depleting agents sometimes raises concern foranticipated toxic side-effects in normal tissues. However, cancer cellsare apparently preferentially vulnerable to ATP depletion, and note thatthe markedly increased tumor-regressing results of PALM-BrPA, whichcombines four ATP-depleting components, is not accompanied by grossdamage to normal tissues, or mortality.

Other ATP Depleters that could Contribute to an ATP-Depleting Regimen

A. Inhibitors of Mitochondrial ATP—There are a number ofmitochondriotoxic small molecules that selectively accumulate in themitochondria of tumor cells, compromise their functional integrity, haveantiproliferative activity, and can cause a significant depletion of ATPpools. F16 is a small molecule that belongs to this group of compoundsthat have cancer cell mitochondria as a selective target (19). Althoughonly tumor growth inhibitory as a single agent in vivo, its selectivityand low toxicity suggests that it could make a substantial contributionto ATP depletion as a component of a necrosis-inducing ATP-depletingcombination (e.g., MAPAL-F16). F16's selectivity appears restricted tobreast cancers. There are other such “selective” mitochondriotoxicmolecules that display greater diversity in accumulating in themitochondria of other types of cancer (20-21), and these should beevaluated in a multi ATP-depleting combination.

B. Inhibitors of Methylthioadenosine phosphorylase (MTAP)—MTAP is animportant intracellular salvage enzyme for adenine (and methionine).Cancer cells that are deficient in MTAP are not able to salvage adenine(and methionine), and are markedly sensitive to inhibitors of the denovo synthesis of adenine nucleotides, especially alanosine which blocksde novo AMP synthesis (22). An inhibitor of MTAP would likely be auseful component of a multi-ATP-depleting combination, because MTAPplays a critical role in recycling adenine moieties fromS-adenosylmethionine, derived originally from ATP, back to adeninenucleotide pools (23).

C. Inhibitors of De Novo Purine Synthesis Other Than6-Methylmercaptopurine riboside (MMPR)—all tumors contain geneticinstability and alterations, including chromosome losses (i.e., genedeletions) and gains (i.e., overexpressed genes). (24) This geneticinstability is reflected in the heterogeneity seen within individualtumors and among tumor of the same type.

Against this background, it is not surprising that MMPR, which requiresmetabiotic activation by phosphorylation via adenosine kinase (25),meets with resistance in cancers devoid of adenosine kinase (26-27).Since MMPR is an important component of MAPAL and PALM, theATP-depleting strategy would not be effective in adenosinekinase-deficient cancer cells. Also, cancer cells with overexpressedmultidrug resistance protein 4, a drug-efflux pump that transports MMPRout of the cell, would reduce the MMPR intracellular concentration, andbe a resistance mechanism (28). Substitution of MMPR in themulti-ATP-depleting regimen by antifolates that target purine synthesisshould be as effective as MMPR, might even be better, and should beevaluated. These novel antifolates include DDATHF (29), Agouron compoundAG 2034 (30), and PDX (31-32).

REFERENCES

-   1. Martin, D. S., Bertino, J. R. and Koutcher, J. A. ATP    depletion+pyrimidine depletion can markedly enhance cancer therapy:    Fresh insight for a new approach. Cancer Res. 60: 6776-6783, 2000.-   2. Shantz, G. D., Smith, C. M., Fontanella, L. J., Lau, H. K. F.,    and Henderson, J. F. Inhibition of purine nucleotide metabolism by    6-methylmercaptopurine ribonucleoside and structurally related    compounds. Cancer Res. 33: 2867-2871, 1973.-   3. Hunting, D. Gowans, B., and Henderson, J. F. Effect of 6-AN on    cell growth, poly (ADP-ribose) synthesis and nucleotide metabolism.    Biochem. Pharmacol. 34: 3999-4003, 1985.-   4. Street, J. C., Mahmoud, V., Ballon, D., Alfieri, A. A., and    Koutcher, J. A. ¹³C and ³¹P NMR investigation of effect of    6-aminonicotinamide on metabolism of RIF-1 tumor cells in vitro. J.    Biol. Chem. 271: 4113-4119, 1996.-   5. Street, J. C., Alfieri, A. A., and Koutcher, J. A. Quantitation    of metabolic and radiobiological effects of 6-aminonicotinamide in    RIF-1 tumor cells in vitro. Cancer Res. 57: 3956-3962, 1997.-   6. Koutcher, J. A., Alfieri, A. A., Matei, C., Zakian, K. I.,    Street, J. C., and Martin, D. S. In vivo 31p NMR detection of    pentose phosphate pathway block and enhancement of radiation    sensitivity with 6-aminonicotinamide. Magn. Reson. Med. 36: 887-892,    1996.-   7. Sweet, S. and Singh, G. Accumulation of human promyelocytic    leukemic (HL-60) cells at two energetic cell cycle checkpoints.    Cancer Res. 55: 5164-5167, 1995.-   8. Nieminen, A. l., Saylor, A. K., Herman, B., and Lemasters, J. J.    ATP depletion rather than mitochondrial depolarization mediates    hepatocyte killing after metabolic inhibition. Am. J. Physiol. 267:    C67-74, 1994.-   9. Martin, D. S., Stolfi, R. L., Sawyer, R. C., Spiegelman, s.,    Casper, E. S., and Young, C. W. Therapeutic utility of utiliying low    doses of N-(phosphonoacetic)-L-aspartic acid in combination with    5-fluorouracil: a murine study with clinical relevance, Cancer Res.    43: 2317-2321, 1983.-   10. Martin, D. S., Purine and Pyrimidine Biochemistry, and some    relevant clinical and preclinical cancer chemotherapy research.    In: G. Powis and R. A. Prough (eds.), Metabolism and Action of    Anti-Cancer Drugs, pp. 91-140. London: Taylor and Francis, 1987.-   11. Eguchi, Y., Shimizu, S., and Tsujimoto, Y. Intracellular ATP    levels determine cell fate by apoptosis or necrosis. Cancer Res.,    57: 1835-1840, 1997.-   12. Leist, M., Single, B., Castoldi, A. F., Kuknle, S., and    Nicotera, P. Intracellular triphosphate (ATP) concentration: A    switch in the decision between apoptosis and necrosis. J. Exp. Med.,    185: 1835-1840, 1997.-   13. Huschtscha, L. I., Anderson, C. E., Bartier, W. A., and    Tattersall, M. H. N. Anti-cancer drugs and apoptosis. In: M. Lavin    and D. Walters (eds.), Programmed Cell Death, the Cellular and    Molecular Biology of Apoptosis, pp. 269-278. New York: Harwood    Academic, 1993.-   14. Koutcher, J. A., Alfieri, A., Stolfi, R. L., Devitt, M. L.,    Colofiore, J. R., Nord, L. D., and Martin, D. S. Potentiation of a    three drug chemotherapy regimen by radiation. Cancer Res. 53:    3518-3823, 1993.-   15. Scudiero, D. A., Monks, A., and Sausville, E. A. Cell line    designation change: Multidrug-resistant cell line in the NCI    anticancer screen. J. Natl. Cancer Inst. 90: 862, 1998.-   16. Tyagic, A. K. and Cooney, D. A. Biomedical pharmacology,    metabolism and mechanism of action of L-alanosine, a novel, natural    antitumor agent. Adv. Pharmacal. Chemother. 20: 69-121, 1984.-   17. Nguygen, B. T., El Sayed, Y. M., and Sadee, W. Interaction among    the distinct effects of adenine and guanine depletion in mouse    lymphoma cells. Cancer Res. 44: 2272-2277, 1984.-   18. Geschwind, J-F. H., Ko, Y. H., Torbenson, M. S., Magee, C., and    Pedersen, P. L. Novel therapy for liver cancer: Direct inhibitor of    ATP production. Cancer Res, 62: 3909-3913, 2002-   19. Fantin, V. R., Berardi, M. J., Scorrano, L., Koromeyer, S. J.,    and Leder, P. A novel mitochondreotoxic small molecule that    selectively inhibit tumor cell growth. Cancer Cell 2: 29-42, 2002-   20. Modica Napolitano, J. S. and Aprille, J. R. Delocalizld    lipophilic cations selectively target the mitochondria of carcinoma    cells. Adv. Drug Deliv. Rev. 49: 63-70, 2001.-   21. Britten, C. D., Rowinsky, E. K., Baker, S. D., Weiss, G. R.,    Smith, L., staphenson, J., Rothenberg, M., Smetzer, L., Cramer, J.,    Collins, W., Von Hoff, D. D., and Eckhardt, S. G. A Phase 1 and    pharmacokinetic study of the mitochondrial-specific chodacyanine dye    analog MKT 011. Clin. Cancer Res. 6: 42-49, 2000.-   22. Yu, J. Alanosine (UCSD). Curr Opin Investig Drugs 2 (11):    1623-30, 2001.-   23. Williams-Ashman, H. G., Seidenfeld, J. and Galletti, P. Trends    in the biochemical pharmacology of 5′-deoxy-5′-methylthioadenosine.    Biochem pharmacal. 31: 277-288, 1982.-   24. Cahill, D. P., Kinzler, K. W., Vogelstein, B., and Lengauer, C.    Genetic instability and Darwinian selection in tumors. Trends    Biachem. Sci. 59-60, 1999.-   25. Martin, D. S. Purine and pyrimidine biochemistry and some    relevant clinical and preclinical cancer chemotherapy research, in    Metabolism and action of Anticancer Drugs (Powis, G. and    Prough, A. R. ds), pp. 91-139, Taylor and Francis, London.-   26. Cory, A. H., and Cory, J. G. Use of nucleoside Kinase deficient    mouse leukemia L1210 cell lines to determine metabolic routes of    activation of antitumor nucleoside analogs. Adv Enzyme Regul 34:    1-12, 1994.-   27. Young, I., Young, G. L., Wiley, J. S., and van der Weyden, M. B.    Nucleoside transport and cytosine arabinoside (ara C) metabolism in    human T lymphoblasts resistant to ara C, thymidine and    6-methyemercap to purine riboside. Eur J Cancer Clin Oncol 21(9):    1077-82, 1985.-   28. Wielinga, P. R., Reid, G., Challa, E. E., van der Heijden, I.,    van Deemter, L., De Haas M., Mol, C., Kuil, A. J., Groeneveld, E.,    Schuetz, J. D., Brouwer, C., De Abreu, R, A., Wijnholds, J.,    Bejnen, J. H., and Borst, P. Thiopurine metabolism and    identification of the Thiopurine metabolites transported by MRP4 and    MRP5 overexpressed in human embryonic kidney cells. Mol. Pharm, 62:    1321-1331, 2002.-   29. Bronder, J. L. and Moran, R. G. antifolates targeting purine    synthesis allow entry of tumor cells into S phase regardless of p53    function. Cancer Res. 62: 5236-5241, 2002.-   30. Bissett, D., Mcleod, H. L., Sheedy, B., Collier, M., Pithavala,    Y., Paradiso, L., Pitsiladas, M. and Cassidy, J. Phase 1    dose-escalation and pharmacokinetic study of a novel folate analogue    A G 2034. Br. J. Cancer 84: 308-312, 2001.-   31. Sirotnak, F. M., De Graw, J. I., Colwell, W. T., and    Piper, J. R. A new analogue of 10-deazaminopterin with markedly    enhanced curative effects against human tumor xenografts in mice.    Cancer Chemother Pharmacal 42: 313-318, 1998.-   32. Krug, L. M., Ng, K. K., Kris, M. G., Miller, V. A., Tong, W.,    Heelan, R. J., Leon, L., Leung, D., Kelly, J., Grant, S. C., and    Sirotnak, F. M. Phase I and pharmacokinetic study of    10-propargyt-10-deazaminopterin a new antifolate. Clin Cancer Res:    3493-3498, 2000.    ATP-Depleting Combination

(i.e., the ATP-depleting combination that, when co-administered with ananticancer agent effective against the target cancer, achievescell-killing levels of ATP in drug-resistant cancer cells with the leasttoxicity to white blood cells and nerves.)

-   1. The initially employed ATP-depleting    agents—6methyl-mercaptopurine riboside (MMPR)+6-aminonicotinamide    (6-AN)—were administered in combination with    N-(phosphonacetyl)-L-aspartic acid (PALA), and received the acronym:    MAP. MAP enhanced anticancer agent activity against drug-resistant    cancer cells.-   2. Subsequently, MMPR and 6-AN were combined with alanosine (AL),    and received the acronym:MAPAL (i.e., MMPR+6-AN+PALA+AL). MAPAL    proved a more effective ATP-depleting agent than MAP. In in vivo    studies in tumor-bearing animals, MAPAL, when co-administered with    anticancer agents, evidenced greater anticancer activity than MAP,    but with occasional evidence of greater myelotoxicity. MAPAL,    nevertheless, is better than MAP.-   3. The combination of PALA+AL+MMPR without 6-AN (acronym: PALM),    when co-administered with anticancer agents, appears to produce the    same or better antitumor activity than MAPAL without this occasional    toxicity. MAPAL needs to be compared with PALM under the same    conditions.-   4. 6-AN inhibits glycolysis (1), a desirable ATP-depleting effect    since the vast majority of solid cancers depend on glycolysis as a    source of ATP (2). 6-AN acts by inhibiting the oxidative portion of    the Pentose Phosphate Pathway (PPP) via inhibition of the second    enzyme of the PPP. DHEA (dehydroepiandrosterone) inhibits the first    enzymes of the oxidated version of the PPP, and, although inferior    as an inhibitor of glycolosis to 6-AN is less toxic than 6-AN. But    since the glycolytic production of ATP receives substrate    contributions (fructose-6-phosphate and glyceraldehyde-3-phosphate)    from the PPP, the oxidative portion of the PPP, DHEA will likely    produce the same inhibition of glycolysis when administered in    combination with an inhibitor of the non-oxidative portion of the    PPP, oxythiamine (OT). Thus, PALM+DHEA+OT; acronym: PALMDOT.

Nevertheless, a greater blockade of the PPP might be accomplished byinhibiting both the first and the second enzymes of the oxidativeportion of PPP with DHEA+6-AN administration together with simultaneousinhibition of the non-oxidative portion of the PPP by OT; acronym:MAPALDOT. (If MAPALDOT is myelosuppressive, it may be made safe by aco-administration of G-CSF while affecting greater ATP depletion thanPALMDOT.) PALMDOT and MAPALDOT need comparison.

Evaluation of the Differences Between ATP-Depleting Regimens of MAPAL,PALM, PALMDOT and MAPALDOT

Tumor Differences—Different tumor types (e.g., breast cancer vs. ovariancancer vs. pancreatic cancer) may differ materially in the quantitativecontribution of ATP they receive from different ATP-producing metabolicpathways (e.g., from glycolysis as compared to de novo purinemetabolism). If the major contribution is, for example, from de novopurine synthesis, then it may be best to employ PALM as theATP-depleting regimen, for PALM has two inhibitors of de novo purinesynthesis, AL (alanosine) and MMPR (6-methyl-mercaptopurine riboside),and no antiglycolysis component. However, while there are exceptions,the vast majority of solid cancers depend on glucose through glycolysisas an energy source, and it would be imprudent to neglect thisinformation. Hence, there should be evaluation of MAPAL, which containsnot only MMPR and AL, but also has 6-AN, a proven inhibitor ofglycolysis via the oxidative portion of the Pentose Phosphate pathway(PPP). But 6-AN may have toxicities that are non-pertinent to the goalof ATP depletion (such as rare neuroparalyis) and, therefore, it may bebest to use DHEA-, a less toxic inhibitor of the oxidative PPP. DHEA maybe inferior to 6-AN as an inhibitor of glycolytic ATP production, butDHEA and 6-AN have not been previously compared in regard to reductionof glycolytic ATP, and the combination of DHEA+an inhibitor for thenon-oxidative portion of the PPP, OT (oxythiamine), has not beenevaluated (in combination) as regards inhibition of glycolytic ATPproduction.

Greater understanding of the above potential interrelationships may beobtained by comparative studies on three different cancers (the humanbreast cancer xenograft resistant to Adriamycin, the NCI/Adr-Res; thecisplatin (DDP)-doxorubicin-resistant human ovarian cancer xenograft,the SKOV-3; and the S2 (TXT), a taxotere (TXT)-resistant humanpancreatic cancer xenograft) as follows:

-   -   1. Controls    -   2. Cisplatin (DDP) MTD    -   3. Doxorubicin (DOX) MTD    -   4. Taxotere (TXT) MTD    -   5. MAP    -   6. MAPAL    -   7. PALM    -   8. PALMDOT    -   9. MAPALDOT    -   10. DDP half-MTD    -   11. DOX half-MTD    -   12. TXT half-MTD    -   13. MAP+DDP half-MTD; +DOX half-MTD; TXT half-MTD    -   14. MAPAL+DDP half-MTD; +DOX half-MTD; +TXT half-MTD    -   15. PALM+DDP half-MTD; DOX half-MTD; +TXT half-MTD    -   16. PALMDOT+DDP half-MTD; +DOX half-MTD; +TXT half-MTD    -   17. MAPALDOT+DDP half-MTD; +DOX half-MTD; +TXT half-MTD        (The above 27 groups (10 tumor-bearing animals per group)        cannot, practically-speaking, be included in a single transplant        experiment, but can be judiciously covered in a number of        smaller experiments.)

Myelotoxicity Differences—None of the above three anticancer agentsalone at half-MTD, nor the various ATP-depleting regimens alone, causemyelosuppression in their tumor-bearing mice, but in combination (as ingroups 13-17) occasionally do, including a rare fatality which can beprevented by either pyruvate administration, or omission of 6-AN, as inPALM. The indirect evidence, (myelosuppression only in combination,prevention by omission of 6-AN, or administration of pyruvate) suggestsan adverse effect to a metabolic step of the glycolysis pathway ineither the progenitor bone marrow cells or the peripheral bloodleucocytes (PBL). Therefore, before treatment, and at intervals (24, 48,72, and 96 hours) after treatment, bone marrow and PBL will be analyzedfor changes in various steps of glycolysis and the PPP. An understandingof the changes may make it possible to identify a biomarker inglycolysis that will predict which tumor-bearing animal (i.e., patient)will undergo severe neutropenia and make possible early G-CSFadministration for prevention as opposed to treatment by G-CSF. Such abiomarker in PBL might be glucose-6-phosphate dehydrogenase, known to beimportant for cell growth and in cell death(3-4).

The elaborate analysis of “best” ATP-depleting regimens, and control ofPBL toxicity, detailed above is warranted by the importance of the drugresistance problem. Combination chemotherapy has been proven to cure theheterogeneity of a few types of cancer (e.g., testicular cancer), sothere is clear evidence that it is possible to cure cancer withchemotherapy. But chemotherapy fails to cure most solid cancers becauseof drug resistance. Since there is strong scientific evidence in supportof the ATP-depleting therapeutic strategy to circumvent drug resistance,the elaborate evaluation as planned above is because a carefulapplication in the clinic of preclinical guidelines should enableclinical validation of the preclinically-proven strategy. Success isimportant, for the circumvention of drug resistance factors will clearthe way for chemotherapeutic cure of many cancers.

REFERENCES

-   1. Street, J. C., Mahmoud, U., Ballon, D., Alfieri, A. A., and    Koutcher, J. A. 13C and 31P NMR investigation of effect of    6-aminnicotinamide on metabolism of RIF-1 tumor cells in vivo. J.    Biol. Chem. 271: 4113-9, 1996.-   2. Dang, C. V. and Semenza, G. L. Oncogenic alterations of    metabolism. Trends Biochem. Sci. 24: 68-92, 1999.-   3. Tian, W-N., Braunstein, L. D., Pang, J., Stuhlmeier, K. M., Xi,    Q-C., Tian, X., and Stanton, R. C. Importance of Glucose-6-phosphate    dehydrogenase activity for cell growth. J. Biol. Chem. 273:    10609-10617, 1998.-   4. Tiam, W-N., Braunstein, L. D., Apse, K., Pang, J., Rose, M.,    Tian, X., and Stanton, R. C. Importance of glucose-6-phosphate    dehydrogenase activity in cell death. Am. J. Physiol. 276 (Cell    Physiol. 45): C1121-C1131, 1999.    An ATP-Depleting Regimen (e.g., MAPAL)+an Anticancer Agent (e.g.,    Taxotere) Induces Permanent Growth Arrest and a Senescence-Like    Phenotype in Advanced Human Breast Cancer Xenografts (MDA-MB-468)    that Results in Cure.

The induction of cell senescence, along with apoptosis, and other typesof cell death (e.g., necrosis, mitotic catastrophe), can be a majorresponse of cancer cells to cytotoxic and cytostatic agents. Thus,treatment conversion of cancer cells to senescent permanentproliferation arrest and subsequent cell death can add an importantdeterminant of effective treatment outcome.

Neither the anticancer agent at MTD, nor the anticancer agent athalf-MTD alone, nor the ATP-depleting regimen alone, effected senescencein the breast tumor xenografts. Therefore, this identification that onlythe combination of the ATP-depleting regimen with a moderate dose ofanticancer agent can induce or facilitate senescence in tumor cells isan important finding for the improvement of cancer therapy.

Present Status of Cancer Chemotherapy and Drug Resistance

Chemotherapy is the primary treatment once cancer becomes systemic(i.e., metastasizes). Most anticancer agents damage DNA in their targetcancer cells, but the post-damage responses of apoptosis, necrosis,mitotic catastrophe and senescence are thwarted in drug-resistant cancercells. The drug resistance factors are multiple, and range frommechanisms that limit the drug-target interaction (e.g., overexpressionof drug efflux pumps, as p-glycoprotein, and intracellular detoxifiers,as glutathione) to genetic disruption of the apoptotic and senescencepathways. Drug-induced senescence, although difficult to induce, isincreasingly being considered an important consequence of effectivetreatment(2,3). Mitotic catastrophe is a form of programmed cell deathwhen the DNA-damaged cells exit a cell cycle arrest and undergo fatalendomitosis(1). It is the contribution of apoptosis to therapy-inducedcell death (the “apoptosis concept” that is considered by most as thepivotal response program in drug-treated tumor cells (4). And necrosis,although occasionally listed as a drug-induced response, is usually notconsidered.

Yet, necrosis is the cell death pathway simultaneously initiated withapoptosis by drug-induced DNA damage that, unlike apoptosis, can berestored to the completion of cell death (5). Necrosis is due to severeATP depletion (15% of normal and below), a cell-killing level preventedfrom attainment by drug-resistant factors (5). However, ATP isnevertheless reduced due to the activation of PARP by anticanceragent-induced DNA damage (5). Thus, unlike “mitotic catastrophe” and thecell death mode of apoptosis, which are initiated but not completed indrug-resistant cancer cells, the necrosis pathway in drug-resistantcells is partially completed (5). The anticancer agent-induced DNAdamage, even though lessened by drug-resistant factors, reduces ATP andthereby “chemosensitizes” the drug-resistant cancer cell for furtherreduction to cell-killing ATP levels by the co-administration of anappropriate ATP-depleting regimen (5). It does not matter whether one ormultiple drug-resistant factors are involved in the limitation of thenecrosis pathway because the treatment to circumvent the drug-resistantfactors is aimed at bringing the product of their collective blockingaction—i.e., the reduced ATP level—to cell-killing levels. The abilityto kill drug-resistant cancer cells by an apoptotic independentmechanism—i.e., necrosis—has been preclinically demonstrated, and isreceiving NCI support for validation of the ATP-depleting strategy byclinical trial.

As noted above, there is growing evidence that senescence of cancercells can be induced following chemotherapy and can contribute to thesuccess of chemotherapy (2,3). It is of great additional interest thatthe same ATP-depleting strategy that enhances cancer cell death byinducing necrosis, also can induce senescence in cancer cells in vivo.(The latter gratifying results require additional confirmation in othertumor models and with other anticancer agents.)

Chemotherapy has cured a few types of human tumors. However, many humanadvanced solid cancers respond poorly to chemotherapy because ofdrug-resistant cells. The ATP-depleting strategy could circumvent thelatter problem, and thereby create the opportunity for chemotherapeuticcure of the more common human malignancies. The belief, and generalconsensus that chemotherapy kills by apoptosis, and therefore that cellsresistant to apoptosis are resistant to drug therapy, neglects thelong-established fact that chemotherapy also kills by necrosis (5-7),and overlooks the evidence that cell death by blocked apoptosis can be“switched” to necrosis (7-9). Overcoming drug resistance is the mostimportant obstacle to the success of chemotherapy in the cure ofadvanced human cancers.

REFERENCES

-   1. King, K. L. and Cidlowski, J. A. Cell cycle and apoptosis: common    pathways to life and death. J. Cell Biochem 58: 175-180, 1995.-   2. Berns, A. Senescence: A companion in chemotherapy? Cancer Cell:    May, 2002; 309-311.-   3. Schmitt, C. A., Fridman, J. S., Yang, M., Lee, S., Baranov, E.,    Hoffman, R. M., and Lowe, S. W. A senescence program controlled by    p53 and p16 ink4a contributed to the outcome of cancer therapy. Cell    109: 335-346, 2002.-   4. Schmitt, C. A. and Lowe, S. W. Apoptosis and chemoresistance in    transgenic cancer models. J. Mol. Med. 80: 137-146, 2002.-   5. Martin, D. S., Bertino, J. R., and Koutcher, J. A. ATP    depletion+pyrimidine depletion can markedly enhance cancer therapy:    Fresh insight for a new approach. Cancer Res. 60: 6776-6783, 2000.-   6. Eguchi, Y., Shimizu, S., and Tsujimoto, Y. Intracellular ATP    levels determine cell fate by apoptosis or necrosis. Cancer Res. 57:    1835-1840, 1997.-   7. Leist, M., Single, B., Castoldo, A. F., Kuknle, S., and    Nicotera, P. Intracellular triphosphate (ATP) concentration: a    switch in the decision between apoptosis and necrosis. J. Exp. Med.    185: 1481-1486, 1997.-   8. Sane, A. T., and Bertrand, R. Caspase inhibition in    camptothecin-treated U-937 cells is completed with a shift from    apoptosis to transient G1 arrest followed by necrotic cell death.    Cancer Res., 59: 3565-3569, 1999.-   9. Lemaire, C., Andreau, K., Souvannavong, K., and Adam, A.

Inhibition of caspase activity induced a switch from apoptosis tonecrosis. FEBS Lett. 425: 266-270, 1998.

1. (canceled)
 2. A composition comprising an effective amount of acombination of ATP-depleting agents at concentrations which deplete theATP level to at least 15% of normal in cancer cells wherein at least oneof the ATP-depleting agents is a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor oran inhibitor of De Novo purine synthesis other than6-Methylmercaptopurine riboside.
 3. The composition of claim 2, whereinsaid composition produces a substantially better effect than acomposition without at least one of the following ATP-depleting agents:a mitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor or an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside.
 4. Thecomposition of claim 2, further comprising a pyrimidine-depleting agentor a pyrimidine antagonist.
 5. The composition of claim 2, furthercomprising an anticancer agent.
 6. The composition of claim 5, whereinthe anticancer agent to which the cancer is sensitive.
 7. Thecomposition of claim 5, wherein the anticancer agent is at approximatelyhalf of the maximum tolerated dose.
 8. The composition of claim 2,wherein the ATP-depleting agents is 6-methylmercaptopurine riboside(MMPR), 6-Aminonicotinamide (6-AN), alanosine (AL) or a combinationthereof.
 9. The composition of claim 8, further comprisingN-(phosphonacetyl)-L-aspartic acid (PALA).
 10. The composition of claim9, further comprising 3-bromopyruvic acid.
 11. The composition of claim2, wherein the ATP-depleting agents is 6-methylmercaptopurine riboside(MMPR), alanosine (AL) or a combination thereof.
 12. The composition ofclaim 11, further comprising N-(phosphonacetyl)-L-aspartic acid (PALA).13. The composition of claim 11, further comprisingdehydroepiandrosterone (DHEA).
 14. The composition of claim 11, furthercomprising oxythiamine (OT).
 15. The composition of claim 11, furthercomprising dehydroepiandrosterone (DHEA) and oxythiamine (OT).
 16. Thecomposition of claim 11, further comprising 6-Aminonicotinomide (6-AN).17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. A methodfor treating a cancer subject comprising administering to the subject acombination of ATP-depleting agents at concentrations which deplete theATP level to at least 15% of normal in cancer cells wherein at least oneof the ATP-depleting agents is a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor oran inhibitor of De Novo purine synthesis other than6-Methylmercaptopurine riboside, wherein said composition produces asubstantially better effect than a composition without at least one ofthe following ATP-depleting agents: a mitochondrial ATP-inhibitor, aglycolytic inhibitor, a methylthioadenosine phosphorylase inhibitor oran inhibitor of De Novo purine synthesis other than6-Methylmercaptopurine riboside.
 22. (canceled)
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled) 33.(canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled)38. (canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled) 42.(canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)
 46. A method fortreating drug-resistant cancer cells comprising contacting the saidcancer with a combination of ATP-depleting agents and an anticanceragent.
 47. The method of claim 46, wherein the dose of said anticanceragent is at approximately half of the maximal tolerated dose.
 48. Themethod of claim 46, wherein the ATP level is depleted to at least 15% ofnormal in cancer cells and at least one of the ATP-depleting agents is amitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor or an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside.
 49. Themethod of claim 46, wherein the ATP level is depleted to at least 15% ofnormal in cancer cells and at least one of the ATP-depleting agents is amitochondrial ATP-inhibitor, a glycolytic inhibitor, amethylthioadenosine phosphorylase inhibitor or an inhibitor of De Novopurine synthesis other than 6-Methylmercaptopurine riboside and saidcomposition produces a substantially better effect than a compositionwithout at least one of the ATP-depleting agents: a mitochondrialATP-inhibitor, a glycolytic inhibitor, a methylthioadenosinephosphorylase inhibitor and an inhibitor of De Novo purine synthesisother than 6-Methylmercaptopurine riboside.
 50. (canceled) 51.(canceled)
 52. (canceled)
 53. (canceled)